Emergence Of Hematopoietic Progenitors Research Articles

BCR::ABL1 -Dependent Promotion of Hematopoietic Stem Cells (HSC) and Progenitors in an Inducible Embryonic Stem Cell (ESC) Model

Our current understanding of the effect of BCR::ABL1 oncogene in leukemia development largely relies on studies using in vitro and in vivo leukemogenesis models. Among them, in vivo models using retrovirus-mediated BCR-ABL gene transfer in murine embryonic stem cell (ESC)-derived hematopoietic cells suggested that the enforced expression of BCR::ABL1 was sufficient to confer them an in vivo long-term repopulation potential. However, in these models, the unique BCR::ABL1 dependence of the generation of this potential has not been shown. Our study aimed at getting a better understanding of the early events regulated by BCR::ABL1 during the course of ESC differentiation without retroviral-mediated gene transfer. The elucidation of these early events could also be of major interest in therapy as generation of bona fide HSCs from normal ESC or iPSC remains a major challenge. To this end, we engineered a unique Tet-ON inducible system in which BCR::ABL1(p210) expression level can be tightly controlled using doxycycline (Dox). As compared to previously reported ESC-derived models, our system contains a single copy of BCR::ABL1 inserted into the murine Col1a1 locus under the control of Tetracycline operator, is sensitive to Dox, leakproof and reversible. The first striking results was the timing of Dox-inducible BCR-ABL expression during the course of ESC differentiation, impacting both the frequency and emergence of hematopoietic progenitors. Indeed, the expression of BCR::ABL1 at day+5 favors significantly the emergence and maintenance of immature hematopoietic progenitors. Importantly, these cells can self-renew only in the presence of Dox. Several hematopoietic stem-like cell lines were established and two of them (HSCL-A and HSCL-C) have been explored in detail. A major BCR-ABL-dependent proliferative ability was documented in both (Figure 1D). Both expressed Lin-Sca-1+c-Kit+CD150+CD48- and eSLAM, corresponding to phenotypically identified adult mouse HSCs. Importantly, these cells exhibited a normal myeloid and erythroid differentiation capacity in CFC assays, giving rise also to CFU-GEMM. We then evaluated the ability of HSCL to self-renew using long-term culture-initiating cell (LTC-IC) assays. To this purpose, EPCR+CD45+/loCD48-CD150+ (ESLAM) cells were sorted at a single cell level and co-cultured on a supportive AFT024 feeder layers for 4 weeks. LTC-IC-derived progenitors were then assessed using CFC assays. The frequency of ESLAM LTC-ICs was 0.53 from adult bone marrow and 0.52 from HSCL lines demonstrating their self-renewal ability. To better characterize HSCL cell lines, we sorted LSK cell population from HSCL#A, HSCL#C, E14.5 fetal liver and murine adult bone marrow and analyzed their transcriptome signatures by RNA-Seq. Unsupervised principal component analysis with the 1333 genes exhibiting the highest variability revealed that HSCL cell lines clustered together and exhibited distinct molecular signature from fetal and adult LSK. This stratification was validated by clustering with the 50 best variable genes. Among them, we found 17 genes highly expressed in HSCL cell lines associated with HSC quiescence (such as NupR1). GSEA analyses confirmed an enrichment of several cell cycle genes as well as genes involved in unfolded protein response (UPR) and autophagy (p <0.001). Thus, we report here for the first time, a unique BCR-ABL inducible ESC-derived hematopoiesis model which demonstrates the ability of BCR-ABL expression to induce a proliferative signal for extended periods of time in highly primitive hematopoietic cells. This novel tool can be used to dissect CML ontogeny both in vitro and in vivo and perform drug screening at the most primitive stem cell level. It can also be of major interest to uncover molecular events and signaling pathways activated during normal ESC-derived hematopoiesis. Such knowledge will open up new avenues to differentiate human iPSC towards bona fide HSCs for potential clinical applications. Figure 1. Establishment of ES cell-derived hematopoietic cell lines expressing BCR::ABLp210.(A) Representation of the timeline of ES cell differentiation protocol and doxycycline induction. (B) HSCL#A cell morphology. (C) MGG staining of HSCL#A cells. (D) Proliferation curves of HSCL#A and HSCL#C cell lines over 40 days in culture. (E)Kinetics of the effect of doxycycline removal on HSCL#A cells cultured on OP9 cells for 4 consecutive days. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Notch and Aryl Hydrocarbon Receptor Signaling Impact Definitive Hematopoiesis from Human Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) stand to revolutionize the way we study human development, model disease, and eventually, treat patients. However, these cell sources produce progeny that retain embryonic and/or fetal characteristics. The failure to mature to definitive, adult‐type cells is a major barrier for iPSC‐based disease modeling and drug discovery. To directly address these concerns, we have developed a chemically defined, serum and feeder‐free–directed differentiation platform to generate hematopoietic stem‐progenitor cells (HSPCs) and resultant adult‐type progeny from iPSCs. This system allows for strict control of signaling pathways over time through growth factor and/or small molecule modulation. Through direct comparison with our previously described protocol for the production of primitive wave hematopoietic cells, we demonstrate that induced HSPCs are enhanced for erythroid and myeloid colony forming potential, and strikingly, resultant erythroid‐lineage cells display enhanced expression of adult β globin indicating definitive pathway patterning. Using this system, we demonstrate the stage‐specific roles of two key signaling pathways, Notch and the aryl hydrocarbon receptor (AHR), in the derivation of definitive hematopoietic cells. We illustrate the stage‐specific necessity of Notch signaling in the emergence of hematopoietic progenitors and downstream definitive, adult‐type erythroblasts. We also show that genetic or small molecule inhibition of the AHR results in the increased production of CD34+CD45+ HSPCs while conversely, activation of the same receptor results in a block of hematopoietic cell emergence. Results presented here should have broad implications for hematopoietic stem cell transplantation and future clinical translation of iPSC‐derived blood cells. Stem Cells 2018;36:1004–1019

Drosophila hematopoiesis under normal conditions and in response to immune stress.

The emergence of hematopoietic progenitors and their differentiation into various highly specialized blood cell types constitute a finely tuned process. Unveiling the genetic cascades that control blood cell progenitor fate and understanding how they are modulated in response to environmental changes are two major challenges in the field of hematopoiesis. In the last 20 years, many studies have established important functional analogies between blood cell development in vertebrates and in the fruit fly, Drosophila melanogaster. Thereby, Drosophila has emerged as a powerful genetic model for studying mechanisms that control hematopoiesis during normal development or in pathological situations. Moreover, recent advances in Drosophila have highlighted how intricate cell communication networks and microenvironmental cues regulate blood cell homeostasis. They have also revealed the striking plasticity of Drosophila mature blood cells and the presence of different sites of hematopoiesis in the larva. This review provides an overview of Drosophila hematopoiesis during development and summarizes our current knowledge on the molecular processes controlling larval hematopoiesis, both under normal conditions and in response to an immune challenge, such as wasp parasitism.

Impaired hematopoietic differentiation of RUNX1-mutated induced pluripotent stem cells derived from FPD/AML patients.

Somatic mutation of RUNX1 is implicated in various hematological malignancies, including myelodysplastic syndrome and acute myeloid leukemia (AML), and previous studies using mouse models disclosed its critical roles in hematopoiesis. However, the role of RUNX1 in human hematopoiesis has never been tested in experimental settings. Familial platelet disorder (FPD)/AML is an autosomal dominant disorder caused by germline mutation of RUNX1, marked by thrombocytopenia and propensity to acute leukemia. To investigate the physiological function of RUNX1 in human hematopoiesis and pathophysiology of FPD/AML, we derived induced pluripotent stem cells (iPSCs) from three distinct FPD/AML pedigrees (FPD-iPSCs) and examined their defects in hematopoietic differentiation. By in vitro differentiation assays, FPD-iPSCs were clearly defective in the emergence of hematopoietic progenitors and differentiation of megakaryocytes, and overexpression of wild-type (WT)-RUNX1 reversed most of these phenotypes. We further demonstrated that overexpression of mutant-RUNX1 in WT-iPSCs did not recapitulate the phenotype of FPD-iPSCs, showing that the mutations were of loss-of-function type. Taken together, this study demonstrated that haploinsufficient RUNX1 allele imposed cell-intrinsic defects on hematopoietic differentiation in human experimental settings and revealed differential impacts of RUNX1 dosage on human and murine megakaryopoiesis. FPD-iPSCs will be a useful tool to investigate mutant RUNX1-mediated molecular processes in hematopoiesis and leukemogenesis.